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Auto Tag: Stress response

April 21, 2020  |  

Lignin catabolic pathways reveal unique characteristics of dye-decolorizing peroxidases in Pseudomonas putida.

Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non-model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin-degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye-decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O2 -dependent ring cleavage strategy was utilized in central pathways to catabolize lignin-derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression-dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn2+ independent lignin depolymerization activity, while Mn2+ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 1012 cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate-limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141-fold increase in DypB activity (56,565?U/l) was achieved, providing novel insights for lignin bioconversion. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.

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April 21, 2020  |  

Deciphering bacterial epigenomes using modern sequencing technologies.

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen-host interactions.

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April 21, 2020  |  

Acid stress response of Staphylococcus xylosus elicits changes in the proteome and cellular membrane.

Coagulase-negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH-values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami.A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH-static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness.This study corroborates mechanisms as previously described for other Gram-positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation.This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci. © 2019 The Society for Applied Microbiology.

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April 21, 2020  |  

Diploid Genome Assembly of the Wine Grape Carménère.

In this genome report, we describe the sequencing and annotation of the genome of the wine grape Carménère (clone 02, VCR-702). Long considered extinct, this old French wine grape variety is now cultivated mostly in Chile where it was imported in the 1850s just before the European phylloxera epidemic. Genomic DNA was sequenced using Single Molecule Real Time technology and assembled with FALCON-Unzip, a diploid-aware assembly pipeline. To optimize the contiguity and completeness of the assembly, we tested about a thousand combinations of assembly parameters, sequencing coverage, error correction and repeat masking methods. The final scaffolds provide a complete and phased representation of the diploid genome of this wine grape. Comparison of the two haplotypes revealed numerous heterozygous variants, including loss-of-function ones, some of which in genes associated with polyphenol biosynthesis. Comparisons with other publicly available grape genomes and transcriptomes showed the impact of structural variation on gene content differences between Carménère and other wine grape cultivars. Among the putative cultivar-specific genes, we identified genes potentially involved in aroma production and stress responses. The genome assembly of Carménère expands the representation of the genomic variability in grapes and will enable studies that aim to understand its distinctive organoleptic and agronomical features and assess its still elusive extant genetic variability. A genome browser for Carménère, its annotation, and an associated blast tool are available at http://cantulab.github.io/data.Copyright © 2019 Minio et al.

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April 21, 2020  |  

Blast Fungal Genomes Show Frequent Chromosomal Changes, Gene Gains and Losses, and Effector Gene Turnover.

Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020  |  

Petunia-and Arabidopsis-Specific Root Microbiota Responses to Phosphate Supplementation

Phosphorus (P) is a limiting element for plant growth. Several root microbes, including arbuscular mycorrhizal fungi (AMF), have the capacity to improve plant nutrition and their abundance is known to depend on P fertility. However, how complex root-associated bacterial and fungal communities respond to various levels of P supplementation remains ill-defined. Here we investigated the responses of the root-associated bacteria and fungi to varying levels of P supply using 16S rRNA gene and internal transcribed spacer amplicon sequencing. We grew Petunia, which forms symbiosis with AMF, and the nonmycorrhizal model species Arabidopsis as a control in a soil that is limiting in plant-available P and we then supplemented the plants with complete fertilizer solutions that varied only in their phosphate concentrations. We searched for microbes, whose abundances varied by P fertilization, tested whether a core microbiota responding to the P treatments could be identified and asked whether bacterial and fungal co-occurrence patterns change in response to the varying P levels. Root microbiota composition varied substantially in response to the varying P application. A core microbiota was not identified as different bacterial and fungal groups responded to low-P conditions in Arabidopsis and Petunia. Microbes with P-dependent abundance patterns included Mortierellomycotina in Arabidopsis, while in Petunia, they included AMF and their symbiotic endobacteria. Of note, the P-dependent root colonization by AMF was reliably quantified by sequencing. The fact that the root microbiotas of the two plant species responded differently to low-P conditions suggests that plant species specificity would need to be considered for the eventual development of microbial products that improve plant P nutrition.

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April 21, 2020  |  

Comparative Genome Characterization of a Petroleum-Degrading Bacillus subtilis Strain DM2.

The complete genome sequence of Bacillus subtilis strain DM2 isolated from petroleum-contaminated soil on the Tibetan Plateau was determined. The genome of strain DM2 consists of a circular chromosome of 4,238,631 bp for 4458 protein-coding genes and a plasmid of 84,240 bp coding for 103 genes. Thirty-four genomic islands coding for 330 proteins and 5 prophages are found in the genome. The DDH value shows that strain DM2 belongs to B. subtilis subsp. subtilis subspecies, but significant variations of the genome are also present. Comparative analysis showed that the genome of strain DM2 encodes some strain-specific proteins in comparison with B. subtilis subsp. subtilis str. 168, such as carboxymuconolactone decarboxylase family protein, gfo/Idh/MocA family oxidoreductases, GlsB/YeaQ/YmgE family stress response membrane protein, HlyC/CorC family transporters, LLM class flavin-dependent oxidoreductase, and LPXTG cell wall anchor domain-containing protein. Most of the common strain-specific proteins in DM2 and MJ01 strains, or proteins unique to DM2 strain, are involved in the pathways related to stress response, signaling, and hydrocarbon degradation. Furthermore, the strain DM2 genome contains 122 genes coding for developed two-component systems and 138 genes coding for ABC transporter systems. The prominent features of the strain DM2 genome reflect the evolutionary fitness of this strain to harsh conditions and hydrocarbon utilization.

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April 21, 2020  |  

A global survey of full-length transcriptome of Ginkgo biloba reveals transcript variants involved in flavonoid biosynthesis

Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts, and 1,270 lncRNAs were predicted by removing redundant reads. Further studies reveal that 7 AS, 5 lncRNA, and 6 fusion gene events were identified in flavonoid biosynthesis. A total of 12 gene modules were revealed to be involved in flavonoid metabolism structural genes and transcription factors by constructing co-expression networks. Weighted gene coexpression network analysis (WGCNA) analysis reveals that some hub genes operate during the biosynthesis by identifying transcription factors (TFs) and structure genes. Seven key hub genes were also identified by analyzing the correlation between gene expression level and flavonoids content. The results highlight the importance of SMRT sequencing of the full-length transcriptome in improving genome annotation and elucidating the gene regulation of flavonoid biosynthesis in G. biloba by providing a comprehensive set of reference transcripts.

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April 21, 2020  |  

The complete genome sequence of Thalassospira indica PB8BT insights into adaptation to the marine environment

Thalassospira indica PB8BT was isolated from the deep water of the Indian Ocean. Here we report the complete genome sequence of type strain PB8BT, which comprises 4,701,725?bp with a G?+?C content of 54.9?mol%. We found that numerous genes related to iron acquisition, resistance, motility and chemotaxis, nitrogen, phosphorus and sulfur metabolism, and stress response. These metabolic features and related genes revealed genetic basis for the adaptation to the marine environment. The genome of T. indica PB8BT will be helpful for further insights into its adaptive evolution and ecological role in marine environment.

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April 21, 2020  |  

Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants.

We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.

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April 21, 2020  |  

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.

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April 21, 2020  |  

Genome analysis of the rice coral Montipora capitata.

Corals comprise a biomineralizing cnidarian, dinoflagellate algal symbionts, and associated microbiome of prokaryotes and viruses. Ongoing efforts to conserve coral reefs by identifying the major stress response pathways and thereby laying the foundation to select resistant genotypes rely on a robust genomic foundation. Here we generated and analyzed a high quality long-read based ~886 Mbp nuclear genome assembly and transcriptome data from the dominant rice coral, Montipora capitata from Hawai’i. Our work provides insights into the architecture of coral genomes and shows how they differ in size and gene inventory, putatively due to population size variation. We describe a recent example of foreign gene acquisition via a bacterial gene transfer agent and illustrate the major pathways of stress response that can be used to predict regulatory components of the transcriptional networks in M. capitata. These genomic resources provide insights into the adaptive potential of these sessile, long-lived species in both natural and human influenced environments and facilitate functional and population genomic studies aimed at Hawaiian reef restoration and conservation.

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April 21, 2020  |  

Associated Bacteria Affect Sexual Reproduction by Altering Gene Expression and Metabolic Processes in a Biofilm Inhabiting Diatom.

Diatoms are unicellular algae with a fundamental role in global biogeochemical cycles as major primary producers at the base of aquatic food webs. In recent years, chemical communication between diatoms and associated bacteria has emerged as a key factor in diatom ecology, spurred by conceptual and technological advancements to study the mechanisms underlying these interactions. Here, we use a combination of physiological, transcriptomic, and metabolomic approaches to study the influence of naturally co-existing bacteria, Maribacter sp. and Roseovarius sp., on the sexual reproduction of the biofilm inhabiting marine pennate diatom Seminavis robusta. While Maribacter sp. severely reduces the reproductive success of S. robusta cultures, Roseovarius sp. slightly enhances it. Contrary to our expectation, we demonstrate that the effect of the bacterial exudates is not caused by altered cell-cycle regulation prior to the switch to meiosis. Instead, Maribacter sp. exudates cause a reduced production of diproline, the sexual attraction pheromone of S. robusta. Transcriptomic analyses show that this is likely an indirect consequence of altered intracellular metabolic fluxes in the diatom, especially those related to amino acid biosynthesis, oxidative stress response, and biosynthesis of defense molecules. This study provides the first insights into the influence of bacteria on diatom sexual reproduction and adds a new dimension to the complexity of a still understudied phenomenon in natural diatom populations.

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April 21, 2020  |  

In-Depth Genomic and Phenotypic Characterization of the Antarctic Psychrotolerant Strain Pseudomonas sp. MPC6 Reveals Unique Metabolic Features, Plasticity, and Biotechnological Potential.

We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments.

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